The present invention relates to Acetyl-COA-carboxylase (ACCase) genes from Candida Albicans (C. albicans) and methods for its expression. The invention also relates to novel hybrid organisms for use in such expression methods.
C. albicans is an important fungal pathogen and the most prominent target organism for antifungal research. ACCase is an enzyme of fatty acid biosynthesis and essential for fungal growth and viability. Inhibitors of the ACCase enzyme should therefore be potent antifungals. The ACCase proteins in all organisms are homologous to each other but they also differ significantly in the amino acid sequence. Because selectivity problems (for example fungal versus human) it is extremely important to optimise potential inhibitor leads directly against the target enzyme (C. albicans) and not against a homologous but non-identical model protein, for example from Saccharomyces cerevisiae (S. Cerevisiae).
We have now successfully cloned the ACCase gene from C. albicans (hereinafter referred to as the C. Albicans ACC1 gene) and elucidated its full length DNA sequence and corresponding polypeptide sequence, as set out in FIGS. 4 and 5 of this application respectively. The coding DNA sequence of the C. Albicans ACC1 gene is 6810 nucleotides in length and the corresponding protein sequence is 2270 amino acids in length. As will be explained below there are two forms of the C. Albicans ACC1 gene, the above numbers relate to the longer version, Met1.
Therefore in a first aspect of the present invention we provide a polynucleotide encoding a C.albicans ACCase gene, in particular the (purified) C. albicans ACC1 gene as set out in FIG. 5 hereinafter. It will be appreciated that the polynucleotide may comprise any of the degenerate codes for a particular amino acid including the use of rare codons. The polynucleotide is conveniently as set out in FIG. 4. It will be apparent from FIG. 4 that the gene is characterised by the start codons Met1 and Met2 (as indicated by the first and second underlined at codons, hereinafter referred to as atg1 and atg2 respectively). Both forms of the gene starting from Met1 and Met2 respectively are comprised in the present invention. The invention further comprises convenient fragments of any one of the above sequences. Convenient fragments may be defined by restriction endonuclease digests of sequence, suitable fragments include a full length C. Albicans ACC1 gene (starting with Met1 or Met2) flanked by unique Stu1 (5xe2x80x2-end)-NotI (3xe2x80x2-end) restriction sites as detailed in FIG. 6.
We also provide a polynucleotide probe comprising any one of the above sequences or fragments together with a convenient label or marker, preferably a non-radioactive label or marker. Following procedures well known in the art, the probe may be used to identify corresponding nucleic acid sequences. Such sequences may be comprised in libraries, such as cDNA libraries. We also provide RNA transcripts corresponding to any of the above C. Albicans ACC1 sequences or fragments.
In a further aspect of the invention we provide a C. albicans ACC1 enzyme, especially the ACC1 enzyme having the polypeptide sequence set out in FIG. 5, in isolated and purified form. This is conveniently achieved by expression of the coding DNA sequence of the C. Albicans ACC1 gene set out in FIG. 4, using methods well known in the art (for example as described in the Maniatis cloning manualxe2x80x94Molecular Cloning: A Laboratory Manual, 2nd Edition 1989, J. Sambrook, E. F. Fritsch and Maniatis). As indicated for FIG. 4 above, the enzyme is characterised by two forms Met1 and Met2. Both form of the enzyme are comprised in the present invention.
The C. Albicans ACC1 enzyme of the present invention is useful as a target in biochemical assays. However, to provide sufficient enzyme for a biochemical assay for C. Albicans ACC1 (for example, for a high throughput screen for enzyme inhibitors) this has to be purified. Two major constraints impair this purification.
1) any new organism will necessitate deviation from published procedures because it will differ in its lysis and protease activity. C. albicans is known to express and secrete many aspartyl proteases.
2) The expression of C. Albicans ACC1 is very low and satisfying purification results can only be achieved if the enzyme is overexpressed.
We have now been able to overcome these problems by controlled overexpression of the C. albicans ACC1 in a Saccharomyces strain. This means that subsequent purification of the enzyme may then for example follow published procedures.
Therefore in a further aspect of the present invention we provide a novel expression system for expression of a C. albicans ACC1 gene which system comprises an S. cerevisiae host strain having a C. albicans ACC1 gene inserted in place of the native ACC1 gene from S. Cerevisiae, whereby the C. albicans ACC1 gene is expressed. Preferred S. cerevisiae strains include JK9-3Daxcex1 and its haploid segregants.
The C. albicans ACC1 gene is preferably over-expressed relative to that as may be achieved by a C. albicans wild type strain, ie under the control of its own ACC1 promoter. Whilst we do not wish to be bound by theoretical considerations, we have achieved approximately 14 fold over-expression relative to the wild-type host S. cervisiae strain JK9-3D. This may be achieved by replacing the C. albicans promoter in the expression construct by a stronger and preferably inducible promoter such as the S. cerevisiae GAL1 promoter.
Controlled overexpression is used to improve expression of a C. albicans polypeptide relative to expression under the control of a C. albicans promoter. In addition using procedures outlined in the accompanying examples we have been able to isolate a fully functional C. albicans ACC1 gene as determined by 100% inhibition by SoraphenA.
The novel expression system is conveniently prepared by transformation of a heterozygous ACC1 deletion strain of a convenient S. cerevisiae host by a convenient plasmid comprising the C. albicans ACC1 gene. Transformation is conveniently effected using methods well known in the art of molecular biology (Ito et al. 1983).
The plasmid comprising the C. albicans ACC1 gene and used to transform a convenient S. cerevisiae host represents a further aspect of the invention. Preferred plasmids for insertion of the C. Albicans ACC1 gene include YEp24, pRS316 and pYES2(Invitrogen).
The heterozygous ACC1 deletion strain of a convenient (diploid) S. cerevisiae host is conveniently achieved by disruption preferably using an antibiotic resistance cassette such as the kanamycin resistance cassette described by Wach et al (Yeast, 1994, 10, 1793-1808).
The expression systems of the invention may be used together with, for example cell growth and enzyme isolation procedures identical to or analogous to those described herein, to provide an acetyl-COA-carboxylase (ACCase) gene from C. albicans in sufficient quantity and with sufficient activity for compound screening purposes.
In a further aspect of the invention we provide the use of an acetyl-COA-carboxylase (ACCase) gene from C. albicans in assays to identify inhibitors of the polypeptide. In particular we provide the their use in pharmaceutical or agrochemical research.
As presented above the C. albicans ACC1 enzyme may be used in biochemical assays to identify agents which modulate the activity of the enzyme. The design and implementation of such assays will be evident to the biochemist of ordinary skill. The enzyme may be used to turn over a convenient substrate whilst incorporating/losing a labelled component to define a test system. Test compounds are then introduced into the test system and measurements made to determine their effect on enzyme activity. Particular assays are those used to identify inhibitors of the enzyme useful as antifungal agents. By way of non-limiting example, the activity of the ACC1 enzyme may be determined by (i) following the incorporation (HCO3, Acetyl-CoA) or loss (ATP) of a convenient label from the relevant substrate (T. Tanabe et al, Methods in Enzymology, 1981, 71, 5-60; M. Matasuhashi, Methods in Enzymology, 1969, 14, 3-16), (ii) following the release of inorganic phosphate from ATP (P. Lanzetta et al, Anal. Biochem. 1979, 100, 95-97), or (iii) following the oxidation of NADH in a coupled assay, for example using either fatty acid synthetase or pyruvate kinase/lactate dehydrogenase enzymes. Convenient labels include carbon14, tritium, phosphorous32 or 33.
Any convenient test compound(s) or library of test compounds may be used. Particular test compounds include low molecular weight chemical compounds (molecular weight less than 1500 daltons) suitable as pharmaceutical agents for human, animal or plant use.
The enzyme of the invention, and convenient fragments thereof may be used to raise antibodies. Such antibodies have a number of uses which will be evident to the molecular biologist of ordinary skill. Such uses include (i) monitoring enzyme expression, (ii) the development of assays to measure enzyme activity and precipitation of the enzyme.
In addition we provide antisense polynucleotides specific for all or a part of an ACC1 polynucleotide of the invention.